Anion exchangers have been used to make purified protein products from milk raw materials such as skim milk and whey. They have also been used to modify the properties of milk by the removal of whey proteins from skim milk.
For example GB 1563990 (1980) discloses a method of preparing a mixture of whey proteins (α-lactalbumin, β-lactoglobulin, serum albumin and some immunoglobulin), now known as whey protein isolate (WPI), by passing either skim milk or milk serum (whey) through a column of silica based anion exchanger, washing the column with water and then eluting and recovering the bound protein. Immunoglobulins remaining in the column effluent were recovered by passing this effluent, at the same pH still, through a column of silica which acts as a cation exchanger and adsorbs the immunoglobulins.
EP 0320152 (1989) also discloses a process which contacts an anion exchanger with whey or liquid whey protein concentrate to adsorb most of the whey proteins and form an effluent rich in immunoglobulins. But in EP 0320152 this effluent is treated differently. It is concentrated by ultrafiltration to prepare an immunoglobulin enriched whey protein concentrate powder (WPC).
Both these references refer to the difficulty of performing ion exchange separation of proteins on an industrial scale because many of the known ion exchangers, particularly those based on cellulose and dextran, have weak mechanical properties which do not lend themselves to large scale use. The authors proposed using anion exchangers with better mechanical properties, particularly ones based on silica and coated with cross-linked polymer film containing quaternary ammonium functional groups, such as QMA Spherosil™ (Rhone-Poulenc). Other anion exchangers mentioned specifically were Q Sepharose™ fast flow (Pharmacia), and Q Trisacryl™ (IBF). However all three of these anion exchangers suffer the disadvantage of being expensive which makes it very difficult to recover whey proteins by ion exchange industrially, for economic reasons.
GB 1563990 also discloses a manner of using the anion exchanger to bring about a more selective separation of the whey proteins. This is achieved by passing the milk (or whey) through two columns filled with anion exchanger and used in tandem. The protein recovered from the first column was very rich in β-lactoglobulin while that recovered from the second column contained α-lactalbumin, serum albumin and small quantities of β-lactoglobulin and immunoglobulins. This is a result of β-lactoglobulin binding more tightly to the anion exchanger than the other whey proteins bind. This has been well documented, particularly for this polymer coated silica based anion exchanger QMA Spherosil™. See J. of Dairy Research, 52, 167–181, 1985.
WO 97/26797 describes processes similar to the above processes, but the processes described use as feedstock a whey protein containing solution (such as a UF retentate or WPC) having a high protein concentration and reduced ionic strength.
U.S. Pat. No. 5,077,067 (1991) further discloses the use of any strongly basic type anion exchanger for the selective and quantitative removal of β-lactoglobulins from starting materials containing whey proteins. The examples cite the use of anion exchangers manufactured from different matrices; a cross-linked agarose matrix, Q Sepharose™ (Pharmacia); a cross-linked dextran matrix, QA Sephadex™ (Pharmacia) and a cross-linked polystyrene matrix, Duolite™ A-101 (Rohm and Haas). However none of these are suitable for large scale industrial use, Q Sepharose™ being expensive and QA Sephadex™ suffering from weak mechanical properties as mentioned above. Duolite™ A-101 suffers the disadvantages of most polystyrene based ion exchangers in that generally they are not suitable for use with proteins. In fact Outlinen et. al. in WO 95/19714 (Example 8) demonstrates that Duolite™ A-101 binds neither α-lactalbumin nor β-lactoglobulin in the pH range 5–8. This is typical of the properties of polystyrene based ion exchangers. So once again the processes disclosed suffer from the lack of an anion exchanger particularly useful for an industrial scale process.
Outinen et. al (Lebensm.-Wiss. U.-Technol, 29, 340–343, 1996) surveyed eleven different anion exchangers from five manufacturers. These exchangers were all quaternary ammonium derivatives of cross-linked polystyrene, matrices chosen for their mechanical and chemical stability, for being macroporous and particularly for their low cost. Porosity was a major consideration as open porosity is necessary to aid protein diffusion into and out of the particles and hence important for protein binding capacity. But only one of these, Diaion™ HPA 75 (Mitsubishi Kasei Corp.) was found to have properties suitable for the selective adsorption of β-lactoglobulin from whey. This type of anion exchanger formed the basis of WO 95/19714 for the separation of β-lactoglobulin from whey and whey protein solutions. However even though Diaion™ HPA 75 has the right mechanical properties and cost structure (about 1/100 the cost of QMA Spherosil™) it still suffers the limitation of low protein capacity when compared to conventional, albeit expensive, anion exchangers for protein adsorption. The optimum load of β-lactoglobulin found for Diaion™ HPA 75 was only 16–20 mg of protein/cm3 of exchanger (Outinen, 1996), whereas it would be advantageous to have operating capacities greater than this.
GB 2188526 (1987) discloses the use of anion exchangers to recover a different proteinaceous material from whey at pH 4–6 or more particularly at pH 4.8–5.0. At this pH most of the proteins in whey are not negatively charged and hence are not adsorbed by an anion exchanger. The proteinaceous material, adsorbed at pH 4.8–5.0 and then eluted, is thought to be a mixture of acidic peptides and proteins. Some of these are believed to be present in milk from which the whey is made, and others produced during the manufacture of the whey by the action of proteolytic enzymes on caseins. Many of them are highly sialylated and/or phosphorylated peptides and proteins and they include a group of minor whey proteins known as proteose-peptones. In the case of rennet and cheese whey the major peptide component of this proteinaceous material is glycomacropeptide (GMP) derived from the enzyme action on kappa-casein. In the case of acid whey the proteinaceous material is a mixture of acidic peptides and proteins other than GMP. GB 2188526 thus provides a process for producing GMP containing other acidic peptides and proteins as minor components (from sweet wheys) or a proteinaceous material composed of acidic peptides and proteins but without GMP present (from acid wheys), these products being substantially free of the whey proteins α-lactalbumin, β-lactoglobulin and serum albumin which are isolated by anion exchange at higher pH. Anion exchangers cited in the examples are QMA Spherosil™ (Rhone-Poulenc), Amberlite IRA 958 (Rohm and Haas) and QA Indion™ now known as QA GibcoCel™ (Life Technologies Ltd, Auckland, New Zealand). The Spherosil suffers from being expensive as outlined earlier, the Amberlite™ IRA 958 is a macroporous acrylic based synthetic resin similar to polystyrene resins mentioned earlier in that it also has low capacity for binding proteins. The QA Indion™ is an industrial grade ion exchanger based on regenerated cellulose and normally suitable for protein adsorption but under the particular conditions cited in GB 2188526 found by us to have low capacity for GMP (3 mg/mL).
GB 2251858 (1992) also describes the preparation of ē-casein glycomacropeptide (GMP) using anion exchangers but differs from GB 2188526 in that the anion exchanger is contacted with the milk raw material containing GMP at a pH≧4 instead of pH 4–6. In the applicants' experience this produces a GMP sub-fraction which is highly glycosylated and particularly acidic. (GMP is a mixture of many different glycoforms of the macropeptide.) Anion exchangers cited in this reference are the previously mentioned QMA Spherosil™ and a Sephadex™ exchanger based on cross-linked dextran which is unsuitable for large scale use.
Processes have thus been disclosed for using anion exchangers to prepare whey protein isolate (WPI) from skim milk or whey (GB 1563990), WPI and an immunoglobulin enriched WPC from whey (EP 0320152), β-lactoglobulin and α-lactalbumin enriched protein isolates (GB 1563990), β-lactoglobulin isolate and an α-lactalbuminl enriched WPC (U.S. Pat. No. 5,077,067 and WO 95/19714) and GMP from rennet or cheese wheys or concentrates (GB 2188526 and GB 2251858).
In those cases where skim milk is treated with the anion exchanger at pH 6.6, it has also been disclosed that the resulting milk, depleted in whey protein (especially in β-lactoglobulin), has enhanced heat stability properties useful for the manufacturing of cheeses (FR 2465422, 1981) and other purposes.
Many different anion exchangers are available for demonstrating these processes on a laboratory scale, but few are really suitable for use in the large scale processing of dairy streams for either technical or economic reasons. This might be one reason why these processes in the main have not been commercially utilised.
One matrix that has proved to be particularly useful in large scale separation and purification of whey proteins is regenerated cellulose which has been hydroxyalkylated and cross-linked. Ion exchangers prepared on this matrix are resistant to attrition, have good protein capacity, high flow properties and are available at relatively low cost.
Examples of such ion exchangers based on a hydroxypropylated and cross-linked regenerated cellulose matrix which are commercially available include the SP, CM, QA, and DEAE derivatives sold as SP GibcoCel™, CM GibcoCel™, QA GibcoCel™ and DEAE GibcoCel™ respectively. These ion exchangers were previously sold under the Indion™ brand name. QA GibcoCel™ and SP GibcoCel™ having a substitution level of the QA or SP groups of up to 1.2 milli-equivalents per dry gram (meq/g) are available. SP GibcoCel™, a cation exchanger, has been widely used, but QA GibcoCel™, an anion exchanger, has only enjoyed limited use industrially.
Levison et al (Chimica Oggi/Chemistry Today, 41–48, November/December 1994) refers to three custom made QA celluloses with substitution levels of 0.74, 0.96 and 1.24 meq/g, and discloses that these had similar protein capacities.
Antal and Micko (Carbohydrate Polymers 19, 167–169, 1992) describe the optimization of the reaction of microcrystalline cellulose with the (3-chloro-2-hydroxypropyl)trimethyl-ammonium chloride. The maximum substitution level of quaternary ammonium groups into the cellulose that they were able to obtain with this reagent was 0.94 meq/g.
The celluloses used by Levison and by Antal are not suitable for repetitive use on a large scale.
With the above background in mind, it is an object of the present invention to provide processes of separating whey proteins from whey protein containing solutions which will go some way towards overcoming the disadvantages of the prior art, or at least to provide the public with a useful choice.